Clinical Science (1991)80,149-154
149
Oral carbidopa has no effect on the renal response to angiotensin I1 in normal man D. W. EADINGTON*, C. P. SWAMSON* AND M. R. LEEt *Medical Renal Unit and ?Clinical Pharmacology Unit, Department of Medicine, Royal Infirmary, Edinburgh, U.K.
(Received 11 April/l7 July 1990; accepted 12 September 1990)
SUMMARY
INTRODUCTION
1. The effect of inhibition of intrarenal dopamine synthesis by carbidopa on the renal response to angiotensin I1 infusion was studied in six healthy salt-loaded volunteers. 2. Subjects received an infusion of angiotensin I1 at two doses (0.5 and 1.0 ng m h - kg-I) on two occasions. Before one study they took a single dose of carbidopa (100 mg) by mouth. 3. The plasma concentrations of angiotensin I1 produced by the infusion were similar on both study days. Angiotensin II infusion reduced urinary dopamine excretion on the control day. Urinary dopamine excretion was undetectable at all times after carbidopa, but carbidopa did not change the basal excretion rate of sodium. Despite inhibition of renal dopamine synthesis, the reductions in both absolute and fractional sodium excretion during the angiotensin I1 infusion were not different from those seen in the control study. 4. The reductions in glomerular filtration rate and effective renal plasma flow which occurred during angiotensin I1 infusion were not modified by pretreatment with carbidopa. 5. The renal response to angiotensin I1 is not modulated either wholly or in part by endogenous intrarenal dopamine levels. The fall in urinary dopamine excretion which occurs during angiotensin I1 infusion is consistent with a modulatory role for tubular reabsorptive capacity in the regulation of proximal tubular dopamine synthesis.
The major intrarenal actions of the octapeptide angiotensin II (ANG 11), vasoconstriction [l]and antinatriuresis [ 2 ] , are the opposite of the effects attributed to the endogenous catecholamine dopamine (DA)which is produced in the proximal tubular cells by the action of dopa decarboxylase (aromatic-L-amino-aciddecarboxylase; EC 4.1.1.28) on L-dopa delivered to the kidney by the systemic circulation [31. When infused at pharmacological doses, DA acts on specific renal DA,-receptors to cause afferent arteriolar vasodilatation, enhanced sodium excretion and an accompanying diuresis 141. A physiological role for endogenous DA in renal sodium handling is suggested by the increased renal DA synthesis which follows both acute and chronic sodium loading [5],the parallel falls in urinary DA output and sodium excretion after chronic inhibition of dopa decarboxylase [ 6 ] , and the blunting of saline-induced natriuresis after carbidopa administration in the dog [7]or dopaminergic blockade in man [8].However, a recent study in man found that carbidopa did not influence the natriuretic response to acute saline loading [9]. There is some evidence that DA interacts with the renin-angiotensin-aldosterone system (RAAS) at several levels. The dopaminergic prodrug y-L-glutamyl-L-dopa (gludopa) inhibits renin secretion despite causing natriuresis and a negative sodium balance [lo], suggesting that the high intrarenal concentrations of DA produced by gludopa may directly inhibit renin release. In addition, the changes in the plasma aldosterone response to exogenous ANG I1 infusion which accompany changes in dietary sodium intake are modulated by both DA infusion 111, 121 and by dopaminergic blockade with metoclopramide [13]. We have therefore examined the effect of inhibition of endogenous DA synthesis on the renal response to low dose ANG I1 infusion in normal volunteers to determine if there is a physiologically important intrarenal interaction between the RAAS and DA.
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Key words: angiotensin 11, dopamine, renal haemodynamics, sodium handling. Abbreviations: ANG 11, angiotensin 11; DA, dopamine; ERPF, effective renal plasma flow; GFR, glomerular filtration rate; PAH, p-aminohippurate; RAAS, renin-angiotensin-aldosteronesystem.
Correspondence: Dr D. W. Eadington, Medical Renal Unit, Royal Infirmary, Lauriston Place, Edinburgh EH3 9YW, U.K.
D. W. Eadington et al.
150 METHODS
\
Subjects
Six healthy normotensive male subjects (mean age 32 years, range 27-39 years) were studied on two occasions at least 1 month apart. The subjects were normal on physical examination, and were taking no medications. All gave fully informed consent. The study was approved by the local Medical Ethical Committee. Their normal diet was supplemented with sodium chloride (‘Slow Sodium’, 200 mmol/day) for 5 days before each study; salt-loading was intended to increase renal sensitivity to exogenous ANG 11, allowing the use of doses of ANG I1 with a negligible systemic pressor effect which would produce increments in plasma ANG I1 levels within the physiological range. The subjects abstained from alcohol and xanthine-containing drinks from 18.00 hours on the day before each study, and fasted from 22.00 hours. Study protocol Subjects collected their urine for 24 h before each study. At 07.00 hours they drank 500 ml of water and attended the laboratory at 07.30, when a plastic cannula was inserted into an antecubital vein in each arm. They remained supine thereafter except when standing to pass urine. Loading doses of p-aminohippurate (PAH; 0.45 g) and polyfructosan S (Inutest; 3.5 g) were given in 100 ml of 0.15 mol/l sodium chloride over 15 min, followed by an infusion of PAH (8.3 g/l) and polyfructosan S ( 10 g/l) in 0.15 mol/l sodium chloride at a rate of 120 ml/h, for estimation of effective renal plasma flow ( E M F ) and glomerular filtration rate (GFR),respectively. A further 200 ml of water was drunk at 07.45 hours and again at 08.1 5 hours, and the bladder was emptied at 08.30 hours. The volume of water drunk after each subsequent micturition was adjusted according to the urine volume and the volumes of blood sampled and fluid infused in order to maintain a stable water balance throughout the study. Blood samples were withdrawn at the beginning and end of timed 30 min clearance periods starting at 09.00 hours. After two baseline periods two urine collections were made during infusion of [Val’IANG I1 amide (Hypertensin; Ciba; 0.1 pg/ml in 0.15 mmol/l sodium chloride) at each of two doses, 0.5 and 1.0 ng min- kg- body weight, followed by two further collections after the ANG I1 infusion had been stopped. Blood pressure and heart rate were monitored at 5 min intervals throughout the study using an automated sphygmomanometer (Sentron; CR Bard Inc., Lombard, IL,U.S.A.). On the ‘active’ day subjects took oral carbidopa (Merck, Sharp and Dohme Ltd, Hoddesdon, Herts, U.K.), 100 mg at 07.00 hours and 50 mg at 10.00 hours, to suppress intrarenal DA production. There were no side-effects from either ANG I1 or carbidopa in any subject.
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’
Sample collection and analytical methods Blood samples were collected into chilled heparinized tubes stored on ice, except those for ANG 11 estimations
which were collected into glass tubes containing 0.5 ml of phenanthroline/ethylenediaminetetra-acetate.The tubes were spun immediately at 4“C, and portions of the plasma were stored at - 70°C. Polyfructosan S and PAH levels in plasma and urine were analysed within 48 h of each study by chemical methods [14, 151. Sodium and potassium concentrations were measured by flame photometry. Plasma renin activity was measured by radioimmunoassay of angiotensin I generated under standard conditions [ 161, with intra- and inter-assay coefficients of variation of 4% and 6%, respectively. Plasma ANG I1 and aldosterone were also measured by radioimmunoassay [17, 181. Portions of urine were acidified (5 mol/l HCI) to prevent oxidation of DA, which was measured as previously described in detail [19]. Briefly, after extraction on to alumina and elution with acetic acid, DA was measured by h.p.1.c. using electrochemical detection with Nmethyldopamine (Epinine; Burroughs Wellcome, Beckenham, Kent, U.K.) as an internal standard. Intra- and inter-assay coefficients of variation were 3.3% and 2. lo/& respectively. The lower detection limit of this assay was 5 ng/ml of urine. Calculations and statistical analysis The results are expressed as means with SEM in parentheses. Mean arterial pressure was estimated as diastolic pressure plus one-third of the pulse pressure. Absolute and fractional renal clearances were calculated using standard formulae. The results from the two baseline periods were not significantly different for any variable, and the means of these two periods were used in the statistical analyses. The influence of time and treatment was examined by analysis of variance for repeated measures [20], followed by paired [-tests using a Bonferroni correction as appropriate. A value of 2 P < 0.05 was accepted as statistically significant. RESULTS Urinary sodium excretion was similar during the 24 h before both study days [control, 370 (37) mmol; carbidopa, 335 (27) mmol]. Mean arterial pressure rose minimally during ANG I1 infusion on both days [control, 91 (2) to 94 (3) mmHg, P = not significant; carbidopa, 89 (2) to 95 (1) mmHg, P < 0.02). Heart rate did not change during ANG I1 infusion on either day. Plasma ANG I1 levels were low at baseline as expected after salt loading (Table l ) , and the increment in ANG I1 levels during infusion was not affected by carbidopa. Plasma renin activity and plasma aldosterone were suppressed at baseline and did not change during ANG I1 infusion. Plasma sodium and potassium and urinary potassium excretion did not change during ANG I1 infusion (data not shown). Urinary DA excretion (Fig. 1) was suppressed to undetectable levels (equivalent to < 0.16 nmol/min) in all subjects throughout the study on the day when carbidopa was administered. On the control day, urinary DA excretion fell in all subjects during ANG I1 infusion [1.23 (0.10) to 1.08 (0.08) nmol/min, P
149
Oral carbidopa has no effect on the renal response to angiotensin I1 in normal man D. W. EADINGTON*, C. P. SWAMSON* AND M. R. LEEt *Medical Renal Unit and ?Clinical Pharmacology Unit, Department of Medicine, Royal Infirmary, Edinburgh, U.K.
(Received 11 April/l7 July 1990; accepted 12 September 1990)
SUMMARY
INTRODUCTION
1. The effect of inhibition of intrarenal dopamine synthesis by carbidopa on the renal response to angiotensin I1 infusion was studied in six healthy salt-loaded volunteers. 2. Subjects received an infusion of angiotensin I1 at two doses (0.5 and 1.0 ng m h - kg-I) on two occasions. Before one study they took a single dose of carbidopa (100 mg) by mouth. 3. The plasma concentrations of angiotensin I1 produced by the infusion were similar on both study days. Angiotensin II infusion reduced urinary dopamine excretion on the control day. Urinary dopamine excretion was undetectable at all times after carbidopa, but carbidopa did not change the basal excretion rate of sodium. Despite inhibition of renal dopamine synthesis, the reductions in both absolute and fractional sodium excretion during the angiotensin I1 infusion were not different from those seen in the control study. 4. The reductions in glomerular filtration rate and effective renal plasma flow which occurred during angiotensin I1 infusion were not modified by pretreatment with carbidopa. 5. The renal response to angiotensin I1 is not modulated either wholly or in part by endogenous intrarenal dopamine levels. The fall in urinary dopamine excretion which occurs during angiotensin I1 infusion is consistent with a modulatory role for tubular reabsorptive capacity in the regulation of proximal tubular dopamine synthesis.
The major intrarenal actions of the octapeptide angiotensin II (ANG 11), vasoconstriction [l]and antinatriuresis [ 2 ] , are the opposite of the effects attributed to the endogenous catecholamine dopamine (DA)which is produced in the proximal tubular cells by the action of dopa decarboxylase (aromatic-L-amino-aciddecarboxylase; EC 4.1.1.28) on L-dopa delivered to the kidney by the systemic circulation [31. When infused at pharmacological doses, DA acts on specific renal DA,-receptors to cause afferent arteriolar vasodilatation, enhanced sodium excretion and an accompanying diuresis 141. A physiological role for endogenous DA in renal sodium handling is suggested by the increased renal DA synthesis which follows both acute and chronic sodium loading [5],the parallel falls in urinary DA output and sodium excretion after chronic inhibition of dopa decarboxylase [ 6 ] , and the blunting of saline-induced natriuresis after carbidopa administration in the dog [7]or dopaminergic blockade in man [8].However, a recent study in man found that carbidopa did not influence the natriuretic response to acute saline loading [9]. There is some evidence that DA interacts with the renin-angiotensin-aldosterone system (RAAS) at several levels. The dopaminergic prodrug y-L-glutamyl-L-dopa (gludopa) inhibits renin secretion despite causing natriuresis and a negative sodium balance [lo], suggesting that the high intrarenal concentrations of DA produced by gludopa may directly inhibit renin release. In addition, the changes in the plasma aldosterone response to exogenous ANG I1 infusion which accompany changes in dietary sodium intake are modulated by both DA infusion 111, 121 and by dopaminergic blockade with metoclopramide [13]. We have therefore examined the effect of inhibition of endogenous DA synthesis on the renal response to low dose ANG I1 infusion in normal volunteers to determine if there is a physiologically important intrarenal interaction between the RAAS and DA.
'
Key words: angiotensin 11, dopamine, renal haemodynamics, sodium handling. Abbreviations: ANG 11, angiotensin 11; DA, dopamine; ERPF, effective renal plasma flow; GFR, glomerular filtration rate; PAH, p-aminohippurate; RAAS, renin-angiotensin-aldosteronesystem.
Correspondence: Dr D. W. Eadington, Medical Renal Unit, Royal Infirmary, Lauriston Place, Edinburgh EH3 9YW, U.K.
D. W. Eadington et al.
150 METHODS
\
Subjects
Six healthy normotensive male subjects (mean age 32 years, range 27-39 years) were studied on two occasions at least 1 month apart. The subjects were normal on physical examination, and were taking no medications. All gave fully informed consent. The study was approved by the local Medical Ethical Committee. Their normal diet was supplemented with sodium chloride (‘Slow Sodium’, 200 mmol/day) for 5 days before each study; salt-loading was intended to increase renal sensitivity to exogenous ANG 11, allowing the use of doses of ANG I1 with a negligible systemic pressor effect which would produce increments in plasma ANG I1 levels within the physiological range. The subjects abstained from alcohol and xanthine-containing drinks from 18.00 hours on the day before each study, and fasted from 22.00 hours. Study protocol Subjects collected their urine for 24 h before each study. At 07.00 hours they drank 500 ml of water and attended the laboratory at 07.30, when a plastic cannula was inserted into an antecubital vein in each arm. They remained supine thereafter except when standing to pass urine. Loading doses of p-aminohippurate (PAH; 0.45 g) and polyfructosan S (Inutest; 3.5 g) were given in 100 ml of 0.15 mol/l sodium chloride over 15 min, followed by an infusion of PAH (8.3 g/l) and polyfructosan S ( 10 g/l) in 0.15 mol/l sodium chloride at a rate of 120 ml/h, for estimation of effective renal plasma flow ( E M F ) and glomerular filtration rate (GFR),respectively. A further 200 ml of water was drunk at 07.45 hours and again at 08.1 5 hours, and the bladder was emptied at 08.30 hours. The volume of water drunk after each subsequent micturition was adjusted according to the urine volume and the volumes of blood sampled and fluid infused in order to maintain a stable water balance throughout the study. Blood samples were withdrawn at the beginning and end of timed 30 min clearance periods starting at 09.00 hours. After two baseline periods two urine collections were made during infusion of [Val’IANG I1 amide (Hypertensin; Ciba; 0.1 pg/ml in 0.15 mmol/l sodium chloride) at each of two doses, 0.5 and 1.0 ng min- kg- body weight, followed by two further collections after the ANG I1 infusion had been stopped. Blood pressure and heart rate were monitored at 5 min intervals throughout the study using an automated sphygmomanometer (Sentron; CR Bard Inc., Lombard, IL,U.S.A.). On the ‘active’ day subjects took oral carbidopa (Merck, Sharp and Dohme Ltd, Hoddesdon, Herts, U.K.), 100 mg at 07.00 hours and 50 mg at 10.00 hours, to suppress intrarenal DA production. There were no side-effects from either ANG I1 or carbidopa in any subject.
’
’
Sample collection and analytical methods Blood samples were collected into chilled heparinized tubes stored on ice, except those for ANG 11 estimations
which were collected into glass tubes containing 0.5 ml of phenanthroline/ethylenediaminetetra-acetate.The tubes were spun immediately at 4“C, and portions of the plasma were stored at - 70°C. Polyfructosan S and PAH levels in plasma and urine were analysed within 48 h of each study by chemical methods [14, 151. Sodium and potassium concentrations were measured by flame photometry. Plasma renin activity was measured by radioimmunoassay of angiotensin I generated under standard conditions [ 161, with intra- and inter-assay coefficients of variation of 4% and 6%, respectively. Plasma ANG I1 and aldosterone were also measured by radioimmunoassay [17, 181. Portions of urine were acidified (5 mol/l HCI) to prevent oxidation of DA, which was measured as previously described in detail [19]. Briefly, after extraction on to alumina and elution with acetic acid, DA was measured by h.p.1.c. using electrochemical detection with Nmethyldopamine (Epinine; Burroughs Wellcome, Beckenham, Kent, U.K.) as an internal standard. Intra- and inter-assay coefficients of variation were 3.3% and 2. lo/& respectively. The lower detection limit of this assay was 5 ng/ml of urine. Calculations and statistical analysis The results are expressed as means with SEM in parentheses. Mean arterial pressure was estimated as diastolic pressure plus one-third of the pulse pressure. Absolute and fractional renal clearances were calculated using standard formulae. The results from the two baseline periods were not significantly different for any variable, and the means of these two periods were used in the statistical analyses. The influence of time and treatment was examined by analysis of variance for repeated measures [20], followed by paired [-tests using a Bonferroni correction as appropriate. A value of 2 P < 0.05 was accepted as statistically significant. RESULTS Urinary sodium excretion was similar during the 24 h before both study days [control, 370 (37) mmol; carbidopa, 335 (27) mmol]. Mean arterial pressure rose minimally during ANG I1 infusion on both days [control, 91 (2) to 94 (3) mmHg, P = not significant; carbidopa, 89 (2) to 95 (1) mmHg, P < 0.02). Heart rate did not change during ANG I1 infusion on either day. Plasma ANG I1 levels were low at baseline as expected after salt loading (Table l ) , and the increment in ANG I1 levels during infusion was not affected by carbidopa. Plasma renin activity and plasma aldosterone were suppressed at baseline and did not change during ANG I1 infusion. Plasma sodium and potassium and urinary potassium excretion did not change during ANG I1 infusion (data not shown). Urinary DA excretion (Fig. 1) was suppressed to undetectable levels (equivalent to < 0.16 nmol/min) in all subjects throughout the study on the day when carbidopa was administered. On the control day, urinary DA excretion fell in all subjects during ANG I1 infusion [1.23 (0.10) to 1.08 (0.08) nmol/min, P
